Chronic treatment with D2-antagonist haloperidol leads to inhibitory/excitatory imbalance in striatal D1-neurons

Striatal dysfunction has been implicated in the pathophysiology of schizophrenia, a disorder characterized by positive symptoms such as hallucinations and delusions. Haloperidol is a typical antipsychotic medication used in the treatment of schizophrenia that is known to antagonize dopamine D2 receptors, which are abundantly expressed in the striatum. However, haloperidol’s delayed therapeutic effect also suggests a mechanism of action that may go beyond the acute blocking of D2 receptors. Here, we performed proteomic analysis of striatum brain tissue and found more than 400 proteins significantly altered after 30 days of chronic haloperidol treatment in mice, namely proteins involved in glutamatergic and GABAergic synaptic transmission. Cell-type specific electrophysiological recordings further revealed that haloperidol not only reduces the excitability of striatal medium spiny neurons expressing dopamine D2 receptors (D2-MSNs) but also affects D1-MSNs by increasing the ratio of inhibitory/excitatory synaptic transmission (I/E ratio) specifically onto D1-MSNs but not D2-MSNs. Therefore, we propose the slow remodeling of D1-MSNs as a mechanism mediating the delayed therapeutic effect of haloperidol over striatum circuits. Understanding how haloperidol exactly contributes to treating schizophrenia symptoms may help to improve therapeutic outcomes and elucidate the molecular underpinnings of this disorder.


Figure S1
-General scheme of the approach adopted in the present study.
Mice were exposed to haloperidol or saline for 30 days, followed by a proteomics analysis of the striatum and a parallel electrophysiology analysis of D2 and D1 neurons in the striatum.All icons were obtained from the Noun Project.(A) Simplified representation of the neuronal system, with the representation of chemical and electrical synapses, where the proteins quantified in this dataset belong mainly to the chemical synapse.(B) Overview of the chemical synapse, where it is visible that many of the quantified proteins belong to the neurotransmitter release cycle and the postsynaptic signal transmission.

Figure S3 -Schematic representation of the excitatory and inhibitory synaptic proteins altered after chronic administration of haloperidol.
Up-regulated proteins in haloperidol-treated mice are depicted in red, while downregulated proteins are depicted in blue.Glutamatergic synapses are the main excitatory synapses in the mammalian brain.Here, glutamate is released and binds to glutamate receptors present in the postsynaptic membrane, such as AMPA receptors, promoting local depolarization of the postsynaptic membrane and, consequently, generating excitatory synaptic currents.After its action, glutamate is removed from the synaptic cleft through the excitatory amino acid 2 (EAA2) present in neurons and astrocytes.In neurons, captured glutamate is recycled, while in astrocytes, glutamate is converted to glutamine, which is subsequently transported to presynaptic (excitatory and inhibitory) neuronal terminals.Once in neurons, glutamine is deaminated by the glutaminase kidney isoform (GLSK) to generate glutamate, which in inhibitory neurons is subsequently catalysed by glutamate decarboxylase 2 (GAD2) to produce inhibitory γ-aminobutyric acid (GABA).In the postsynaptic excitatory neuron, PRKCA-binding protein (PICK1) interacts with glutamate ionotropic receptor AMPA type 2 (GluR2) through its PDZ domain, promoting the internalization of AMPA receptors containing the GluR2 subunit.This internalization is regulated by tetraspanin-7 (TSN7) which interacts with the PDZ domain of PICK1, preventing its interaction with GluR2 receptors and their internalization.At inhibitory synapses, after being released into the synaptic cleft, GABA is removed by GABA transporters and, once inside the presynaptic inhibitory neuron, is metabolized by 4-aminobutyrate aminotransferase (GABT).From this reaction, glutamate and succinic semialdehyde are produced.The succinic semialdehyde can be further oxidized to become succinic acid, which enters the citric acid cycle and produces more glutamate.Glutamate can therefore be converted to GABA, as explained above.

Figure S4. Proteomic analysis revealed a total of 405 proteins significantly altered upon chronic exposure to haloperidol.
Volcano plot of the 1482 proteins detected by proteomic analysis.Proteins statistically altered (p<0.1)upon chronic exposure to haloperidol are above the dashed line (405 proteins).Proteins involved in regulating neuronal excitability are highlighted in the volcano plot and further depicted in the violin plots (reduced expression of SCN2B in the haloperidol group, and increased expression of AT1A1, AT1A2, AT1A3, and AT2B4 in the haloperidol group).
SCN2B is a regulatory subunit of voltage-gated sodium channels, which are responsible for the initiation and propagation of action potentials in neurons.The beta-2 subunit modulates the activity of sodium channels by increasing their cell surface expression, slowing their inactivation, and shifting their voltage dependence 2,3 .A reduction in the levels of SCN2B will thus cause a decrease in the cell surface expression of sodium channels, which can decrease neuronal excitability by decreasing the number of channels available for activation.AT2B4 is the plasma membrane calcium-transporting ATPase 4 (PMCA4), which is a protein that pumps calcium ions out of cells, thereby regulating calcium signalling and homeostasis.PMCA4 is widely expressed in many tissues, including the brain, and has been implicated in a variety of cellular processes, including neurotransmitter release, neuronal excitability, and synaptic plasticity [4][5][6] .The role of PMCA4 in regulating neuronal excitability is complex and depends on various factors, such as the specific neuronal population and the cellular context, hence, more research is needed to fully understand the role of PMCA4 in regulating neuronal excitability in different cellular and physiological contexts.Furthermore, we found increased expression of ATP1A1, ATP1A2 and ATP1A3, three sodium/potassium-transporting ATPase subunits that help to maintain the resting membrane potential of neurons by actively transporting sodium ions out of the cell and potassium ions into the cell.An increase in the activity of ATP1A1, ATP1A2 and ATP1A3 can decrease neuronal excitability by hyperpolarizing the cell membrane and increasing the threshold for action potential generation.This is because the outward movement of sodium ions and inward movement of potassium ions generated by ATP1A2/ATP1A3 activity leads to a net negative charge inside the cell, making it more difficult for the membrane potential to depolarize and reach the threshold for action potential firing.
Lastly, several proteins related to mitochondrial metabolism were also found to be changed, which can indirectly influence neuronal excitability.Changes in mitochondrial respiration, reactive oxygen species (ROS) production, and ATP production, can alter the availability of ATP, a key energy source for cellular processes, including ion channel activity and neurotransmitter release.For example, reduced ATP production due to impaired mitochondrial metabolism can lead to decreased activity of ATP-dependent potassium channels and increased activity of voltage-gated calcium channels, leading to an increase in neuronal excitability.Conversely, increased ATP production due to enhanced mitochondrial metabolism can lead to increased activity of ATP-dependent potassium channels and decreased activity of voltage-gated calcium channels, leading to a decrease in neuronal excitability.
A list of all proteins detected by our proteomic analysis can be found in Supplementary Table S1.Furthermore, proteins significantly altered upon chronic exposure to haloperidol can be easily found in another table (Supplementary Table S3) where proteins are ordered by their p values relative to control.